hnRNP A1 and hnRNP C associate with miR-17 and miR-18 in thyroid cancer cells.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are essential players in the regulation of gene expression. The majority of the twenty different hnRNP proteins act through the modulation of pre-mRNA splicing. Most have been shown to regulate the expression of critical genes for the progression of tumorigenic processes and were also observed to be overexpressed in several types of cancer. Moreover, these proteins were described as essential components for the maturation of some microRNAs (miRNAs). In the human genome, over 70% of miRNAs are transcribed from introns; therefore, we hypothesized that regulatory proteins involved with splicing could be important for their maturation. Increased expression of the miR-17-92 cluster has already been shown to be related to the development of many cancers, such as thyroid, lung, and lymphoma. In this article, we show that overexpression of hnRNP A1 and hnRNP C in BCPAP thyroid cancer cells directly affects the expression of miR-17-92 miRNAs. Both proteins associate with the 5'-end of this cluster, strongly precipitate miRNAs miR-17 and miR-18a and upregulate the expression of miR-92a. Upon overexpression of these hnRNPs, BCPAP cells also show increased proliferation, migration, and invasion rates, suggesting upregulation of these proteins and miRNAs is related to an enhanced tumorigenic phenotype.

Thyroid and Breast Cancer in 2 Sisters With Monoallelic Mutations in the Ataxia Telangiectasia Mutated (ATM) Gene.

The presence of a bidirectional risk for metachronous carcinomas among women with thyroid and breast cancer is well established. However, the underlying risk factors remain poorly understood. Two sisters developed papillary thyroid cancer (PTC) at age 32 and 34 years, followed by ductal carcinoma of the breast at 44 and 42 years. The 2 children of the younger sister developed ataxia-telangiectasia; the son also developed lymphoblastic lymphoma and his sister died secondary to acute lymphoblastic leukemia (ALL). They were found to be compound heterozygous for ataxia telangiectasia mutated (ATM) gene mutations (c.3848T>C, p.L1283P; and c.802C>T, p.Q268X). Exome sequencing of the 2 sisters (mother and aunt of the children with ataxia-telangiectasia) led to the detection of the pathogenic monoallelic ATM mutation in both of them (c.3848T>C; minor allele frequency [MAF] < 0.01) but detected no other variants known to confer a risk for PTC or breast cancer. The findings suggest that monoallelic ATM mutations, presumably in conjunction with additional genetic and/or nongenetic factors, can confer a risk for developing PTC and breast cancer.

Muscle Stem Cell Function Is Impaired in ß2-Adrenoceptor Knockout Mice.

Knockout (ko) mice for the ß2 adrenoceptor (Adrß2) have impaired skeletal muscle regeneration, suggesting that this receptor is important for muscle stem cell (satellite cell) function. Here, we investigated the role of Adrß2 in the function of satellite cells from ß2ko mice in the context of muscle regeneration, through in vivo and in vitro experiments. Immunohistochemical analysis showed a significant reduction in the number of self-renewed Pax7+ satellite cells, proliferating Pax7+/MyoD+ myogenic precursor cells, and regenerating eMHC+ myofibers in regenerating muscle of ß2ko mice at 30, 3, and 10 days post-injury, respectively. Quiescent satellite cells were isolated by fluorescence-activated cell sorting, and cell cycle entry was assessed by EdU incorporation. The results demonstrated a lower number of proliferating Pax7+/EdU+ satellite cells from ß2ko mice. There was an increase in the gene expression of the cell cycle inhibitor Cdkn1a and Notch pathway components and the activation of Notch signaling in proliferating myoblasts from ß2ko mice. There was a decrease in the number of myogenin-positive nuclei in myofibers maintained in differentiation media, and a lower fusion index in differentiating myoblasts from ß2ko mice. Furthermore, the gene expression of Wnt/ß-catenin signaling components, the expression of nuclear ß-catenin and the activation of Wnt/ß-catenin signaling decreased in differentiating myoblasts from ß2ko mice. These results indicate that Adrß2 plays a crucial role in satellite cell self-renewal, as well as in myoblast proliferation and differentiation by regulating Notch and Wnt/ß-catenin signaling, respectively.

Functional Toll-like Receptor 4 Overexpression in Papillary Thyroid Cancer by MAPK/ERK-Induced ETS1 Transcriptional Activity.

Emerging evidence suggests that unregulated Toll-like receptor (TLR) signaling promotes tumor survival signals, thus favoring tumor progression. Here, the mechanism underlying TLR4 overexpression in papillary thyroid carcinomas (PTC) mainly harboring the BRAFV600E mutation was studied. TLR4 was overexpressed in PTC compared with nonneoplastic thyroid tissue. Moreover, paired clinical specimens of primary PTC and its lymph node metastasis showed a significant upregulation of TLR4 levels in the metastatic tissues. In agreement, conditional BRAFV600E expression in normal rat thyroid cells and mouse thyroid tissue upregulated TLR4 expression levels. Furthermore, functional TLR4 expression was demonstrated in PTC cells by increased NF-κB transcriptional activity in response to the exogenous TLR4-agonist lipopolysaccharide. Of note, The Cancer Genome Atlas data analysis revealed that BRAFV600E-positive tumors with high TLR4 expression were associated with shorter disease-free survival. Transcriptomic data analysis indicated a positive correlation between TLR4 expression levels and MAPK/ERK signaling activation. Consistently, chemical blockade of MAPK/ERK signaling abrogated BRAFV600E-induced TLR4 expression. A detailed study of the TLR4 promoter revealed a critical MAPK/ERK-sensitive Ets-binding site involved in BRAFV600E responsiveness. Subsequent investigation revealed that the Ets-binding factor ETS1 is critical for BRAFV600E-induced MAPK/ERK signaling-dependent TLR4 gene expression. Together, these data indicate that functional TLR4 overexpression in PTCs is a consequence of thyroid tumor-oncogenic driver dysregulation of MAPK/ERK/ETS1 signaling.Implications: Considering the participation of aberrant NF-κB signaling activation in the promotion of thyroid tumor growth and the association of high TLR4 expression with more aggressive tumors, this study suggests a prooncogenic potential of TLR4 downstream signaling in thyroid tumorigenesis. Mol Cancer Res; 16(5); 833-45. ©2018 AACR.

miR18a and miR19a Recruit Specific Proteins for Splicing in Thyroid Cancer Cells.

BACKGROUND: Thyroid cancer is one of the most frequent types of endocrine cancers. In most cases, thyroid cancers are caused by deregulated miRNA expression, especially involving the miR17-92 cluster. miR17-92 transcription is altered in several different tumor types including lymphoma, leukemia, and of the breast and thyroid. As an intronic cluster, miR17-92 must be processed during splicing and therefore interaction between microprocessor and spliceosome machineries is of major importance in understanding its expression. MATERIALS AND METHODS: We investigated the protein composition of spliceosomes assembled on pre-RNAs containing intronic miR18a and miR19a, components of the miR17-92 cluster, using mass spectrometry. RESULTS: Interestingly, we observed that proteins associated with intronic miR18a and miR19a are cell-specific, and are similar for both miRNAs analyzed. The only exception is the group of heterogeneous nuclear proteins that are commonly recruited by different cells. CONCLUSION: miRNA processing depends on cell-specific proteins and heterogeneous nuclear proteins have a general role in miRNA processing from introns.

Morphometric information to reduce the semantic gap in the characterization of microscopic images of thyroid nodules.

BACKGROUND: The analyses of several systems for medical-imaging processing typically support the extraction of image attributes, but do not comprise some information that characterizes images. For example, morphometry can be applied to find new information about the visual content of an image. The extension of information may result in knowledge. Subsequently, results of mappings can be applied to recognize exam patterns, thus improving the accuracy of image retrieval and allowing a better interpretation of exam results. Although successfully applied in breast lesion images, the morphometric approach is still poorly explored in thyroid lesions due to the high subjectivity thyroid examinations. OBJECTIVE: This paper presents a theoretical-practical study, considering Computer Aided Diagnosis (CAD) and Morphometry, to reduce the semantic discontinuity between medical image features and human interpretation of image content. METHOD: The proposed method aggregates the content of microscopic images characterized by morphometric information and other image attributes extracted by traditional object extraction algorithms. This method carries out segmentation, feature extraction, image labeling and classification. Morphometric analysis was included as an object extraction method in order to verify the improvement of its accuracy for automatic classification of microscopic images. RESULTS: To validate this proposal and verify the utility of morphometric information to characterize thyroid images, a CAD system was created to classify real thyroid image-exams into Papillary Cancer, Goiter and Non-Cancer. Results showed that morphometric information can improve the accuracy and precision of image retrieval and the interpretation of results in computer-aided diagnosis. For example, in the scenario where all the extractors are combined with the morphometric information, the CAD system had its best performance (70% of precision in Papillary cases). CONCLUSION: Results signalized a positive use of morphometric information from images to reduce semantic discontinuity between human interpretation and image characterization.

Integrated Analysis of Thyroid Cancer Public Datasets Reveals Role of Post-Transcriptional Regulation on Tumor Progression by Targeting of Immune System Mediators.

Papillary thyroid carcinoma (PTC) is a well-differentiated thyroid tumor that accounts for approximately 80% of thyroid cancer cases. On other hand, anaplastic thyroid carcinoma (ATC) is a less frequent, but aggressive subtype, with poor prognosis. MicroRNAs (miRNAs), small non-coding RNAs, have emerged as potent post-transcriptional regulators of gene expression, which modulate the expression of cancer-related genes. Computational analyses estimate that a single miRNA may modulate hundreds of mRNA targets and, at the same time, cooperate with others to regulate one single mRNA transcript. Due to the large number of predicted targets and possible interactions, only a small number of miRNAs have characterized biological roles, and the panorama of miRNA-mediated regulation in thyroid cancer remains to be understood. Taking into consideration the large amount of gene expression data deposited in public databases we aligned miRNA target prediction and gene expression data from public PTC and ATC datasets to construct a network of post-transcriptional regulation in thyroid cancer. After a gene set enrichment analysis we identified signaling pathways and biological processes potentially modulated by the miRNAs deregulated in PTC and ATC. Our results show miRNA-mRNA interaction that could contribute with the de-regulation of key tumor-host mediators, such as extra-cellular matrix molecules, interleukins and interleukin receptors, which could drive a more aggressive behavior and tumor progression. Moreover, our analysis through The Cancer Genome Atlas (TCGA) database revealed that aberrant expression of ECM and cytokines genes is frequent in PTC and is associated with aggressive behavior and decreased overall survival rate. In conclusion, we shed light on the post-transcriptional regulation of gene expression in differentiated and undifferentiated thyroid cancers, revealing a potential role of miRNAs in modulation of tumor-host interaction molecules, particularly ECM molecules and immune system mediators, which could stimulate crosstalk between tumors and the immune system to generate a more aggressive behavior. We propose a novel putative miRNA:mRNA network that could lead to a new path toward functional studies.

Regulation of thyroid oxidative state by thioredoxin reductase has a crucial role in thyroid responses to iodide excess.

The phenomenon that supraphysiological doses of iodide (I(-)) temporarily inhibit thyroid hormone synthesis is known as thyroid iodide autoregulation. Recovery of thyroid function has been attributed to sodium-iodide symporter (NIS) inhibition, but the diversity of available data makes it difficult to reach definitive conclusions. Iodide excess induces reactive oxygen species production and cell toxicity. However, the roles of the oxidative state of the cell and antioxidant selenoproteins in I(-) autoregulation have never been explored. Here we analyze the effects of high I(-) doses in rat thyroids and in PCCl3 cells in the period comprising I(-) autoregulation (i.e. 0-72 h after I(-) administration), focusing on NIS expression, redox state, and the expression and activity of selenoproteins. Our results show that NIS mRNA inhibition by I(-) does not occur at the transcriptional level, because neither NIS promoter activity nor Pax8 expression or its binding to DNA was modulated. Because I(-) uptake was inhibited much earlier than NIS protein, and no effect was observed on its subcellular localization, we suggest that I(-) is inhibiting NIS in the plasma membrane. The increased reactive oxygen species production leads to an increase in thioredoxin reductase mRNA levels and enzyme activity, which reduces the oxidative stress. Inhibition of thioredoxin reductase at either gene expression or activity levels prevented NIS recovery, thus illustrating a new role played by this selenoprotein in the regulation of cell homeostasis and consequently in I(-) autoregulation.

Expression of SMAD proteins, TGF-beta/activin signaling mediators, in human thyroid tissues.

OBJECTIVE: To investigate the expression of SMAD proteins in human thyroid tissues since the inactivation of TGF-beta/activin signaling components is reported in several types of cancer. Phosphorylated SMAD 2 and SMAD3 (pSMAD2/3) associated with the SMAD4 induce the signal transduction generated by TGF-beta and activin, while SMAD7 inhibits this intracellular signaling. Although TGF-beta and activin exert antiproliferative roles in thyroid follicular cells, thyroid tumors express high levels of these proteins. MATERIALS AND METHODS: The protein expression of SMADs was evaluated in multinodular goiter, follicular adenoma, papillary and follicular carcinomas by immunohistochemistry. RESULTS: The expression of pSMAD2/3, SMAD4 and SMAD7 was observed in both benign and malignant thyroid tumors. Although pSMAD2/3, SMAD4 and SMAD7 exhibited high cytoplasmic staining in carcinomas, the nuclear staining of pSMAD2/3 was not different between benign and malignant lesions. CONCLUSIONS: The finding of SMADs expression in thyroid cells and the presence of pSMAD2/3 and SMAD4 proteins in the nucleus of tumor cells indicates propagation of TGF-beta/activin signaling. However, the high expression of the inhibitory SMAD7, mostly in malignant tumors, could contribute to the attenuation of the SMADs antiproliferative signaling in thyroid carcinomas.

Expression of SMAD proteins, TGF-beta/activin signaling mediators, in human thyroid tissues / Expressão de proteínas SMAD, mediadores da sinalização de TGF-beta/activina, em tecidos de tiroide humana

OBJECTIVE: To investigate the expression of SMAD proteins in human thyroid tissues since the inactivation of TGF-β/activin signaling components is reported in several types of cancer. Phosphorylated SMAD 2 and SMAD3 (pSMAD2/3) associated with the SMAD4 induce the signal transduction generated by TGF-β and activin, while SMAD7 inhibits this intracellular signaling. Although TGF-β and activin exert antiproliferative roles in thyroid follicular cells, thyroid tumors express high levels of these proteins. MATERIALS AND METHODS: The protein expression of SMADs was evaluated in multinodular goiter, follicular adenoma, papillary and follicular carcinomas by immunohistochemistry. RESULTS: The expression of pSMAD2/3, SMAD4 and SMAD7 was observed in both benign and malignant thyroid tumors. Although pSMAD2/3, SMAD4 and SMAD7 exhibited high cytoplasmic staining in carcinomas, the nuclear staining of pSMAD2/3 was not different between benign and malignant lesions. CONCLUSIONS: The finding of SMADs expression in thyroid cells and the presence of pSMAD2/3 and SMAD4 proteins in the nucleus of tumor cells indicates propagation of TGF-β/activin signaling. However, the high expression of the inhibitory SMAD7, mostly in malignant tumors, could contribute to the attenuation of the SMADs antiproliferative signaling in thyroid carcinomas.

OBJETIVO: Investigar a expressão de proteínas SMAD em tecidos de tiroide humana desde que a inativação dos componentes da sinalização de TGF-β/activina é relatada em diversos tipos de câncer. SMAD 2 e SMAD3 fosforilados (pSMAD2/3) associados com SMAD4 induzem a transmissão do sinal gerado por TGF-β e activina, enquanto SMAD7 inibe essa sinalização intracelular. Embora TGF-β e activina exerçam efeitos antiproliferativos nas células foliculares da tiroide, tumores de tiroide expressam altos níveis dessas proteínas. MATERIAIS E MÉTODOS: A expressão proteica de SMADs foi avaliada em bócio multinodular, adenoma folicular, carcinomas papilífero e folicular por imuno-histoquímica. RESULTADOS: A expressão de pSMAD2/3, SMAD4 e SMAD7 foi observada tanto em tumores benignos como malignos da tiroide. Embora pSMAD2/3, SMAD4 e SMAD7 exibissem alta positividade citoplasmática em carcinomas, a positividade nuclear de pSMAD2/3 não foi diferente entre lesões benignas e malignas da tiroide. CONCLUSÕES: O achado da expressão de SMADs em células tiroidianas e a presença das proteínas pSMAD2/3 e SMAD4 no núcleo de células tumorais indicam propagação da sinalização TGF-β/activina. Contudo, a alta expressão de SMAD7 inibitório, principalmente em tumores malignos, poderia contribuir para atenuação da sinalização antiproliferativa de SMADs em carcinomas de tiroide.

Endogenous expression of Hras(G12V) induces developmental defects and neoplasms with copy number imbalances of the oncogene.

We developed mice with germline endogenous expression of oncogenic Hras to study effects on development and mechanisms of tumor initiation. They had high perinatal mortality, abnormal cranial dimensions, defective dental ameloblasts, and nasal septal deviation, consistent with some of the features of human Costello syndrome. These mice developed papillomas and angiosarcomas, which were associated with Hras(G12V) allelic imbalance and augmented Hras signaling. Endogenous expression of Hras(G12V) was also associated with a higher mutation rate in vivo. Tumor initiation by Hras(G12V) likely requires augmentation of signal output, which in papillomas and angiosarcomas is achieved via increased Hras-gene copy number, which may be favored by a higher mutation frequency in cells expressing the oncoprotein.

Notch activation is associated with tetraploidy and enhanced chromosomal instability in meningiomas.

The Notch signaling cascade is deregulated in diverse cancer types. Specific Notch function in cancer is dependent on the cellular context, the particular homologs expressed, and cross-talk with other signaling pathways. We have previously shown that components of the Notch signaling pathway are deregulated in meningiomas. However, the functional consequence of abnormal Notch signaling to meningiomas is unknown. Here, we report that exogenous expression of the Notch pathway effector, HES1, is associated with tetraploid cells in meningioma cell lines. Activated Notch1 and Notch2 receptors induced endogenous HES1 expression and were associated with tetraploidy in meningiomas. Tetraploid meningioma cells exhibited nuclear features of chromosomal instability and increased frequency of nuclear atypia, such as multipolar mitotic spindles and accumulation of cells with large nuclei. FACS-sorted tetraploid cells are viable but have higher rates of spontaneous apoptosis when compared with diploid cells. We have used spectral karyotyping to show that, in contrast to diploid cells, tetraploid cells develop a higher number of both numerical and structural chromosomal abnormalities. Our findings identify a novel function for the Notch signaling pathway in generating tetraploidy and contributing to chromosomal instability. We speculate that abnormal Notch signaling pathway is an initiating genetic mechanism for meningioma and potentially promotes tumor development.